Abstract The overarching goals of the present proposal are to develop first-in-class multimeric HIV-1 integrase (IN) inhibitors (MINIs) for their future clinical development and to exploit these compounds as powerful investigational tools for HIV-1 molecular biology to uncover critical molecular interactions during maturation. Because of their unique mode of action, MINIs are expected to potently inhibit all drug resistant viral phenotypes in the clinic, which continually evolve in response to currently used ARTs. By rationally modifying archetypal, multifunctional quinoline-based allosteric IN inhibitors (ALLINIs), we have developed highly potent pyridine-based MINIs, which are highly selective for inducing hyper-multimerization of IN. Our SAR studies have been critical for understanding the antiviral mode of action of these inhibitors and allowed us to clearly delineate the significance of HIV-1 IN multimerization as a novel, attractive therapeutic target. We have shown this hyper-multimerization of IN occurs in viral particles during maturation, which in turn impairs IN binding to the viral RNA genome and results in eccentric, non-infectious virions with ribonucleoprotein complexes being displaced outside of the protective capsid core. In addition, our rational design approach enabled us to develop a lead compound, MINI KF116, with a markedly enhanced genetic barrier to resistance compared with its ALLINI counterparts. In particular, KF116 is fully active against the HIV-1 variant with an A128T IN substitution, which confers resistance to the majority of archetypal ALLINIs. Instead, triple (T124N/V165I/T174I) IN substitutions, which significantly compromise viral replication even with a compensatory V165I mutation, are necessary to confer resistance to KF116. Collectively, our findings argue that pyridine-based KF116 is as an excellent platform for the development of second generation MINIs. Our future work will extend these studies and build on our exciting new preliminary results, which show that MINIs/ALLINIs exhibit striking preference for full-length wild type IN tetramers. Specifically, we found that inhibitor binding to the catalytic core domain (CCD) dimer is not sufficient for its activity and that the CCD-inhibitor-C-terminal domain interactions between adjoining full-length IN tetramers are necessary to induce hyper-multimerization of IN. Accordingly, we propose to dissect unique structural features of IN tetramers as authentic targets for MINIs and ALLINIs (aim 1) and utilize this information to optimize second generation inhibitors for their future clinical development (aim 2). The proposed studies are highly complementary to and will synergize with very active ongoing efforts in the pharmaceutical industry to translate the first-in-class MINIs/ALLINIs into the clinic with the ultimate goal of delivering safer next generation therapeutics.